We propose to generate the first high-resolution, comprehensive view of the epigenetic landscape of subtelomeres, the most variable and recombination-prone regions of the human genome. Subtelomeric genes vary markedly in copy number, location, and sequence context among individuals. To fully understand the biological significance of these fast-evolving regions, we will characterize the epigenetic context in which subtelomeric genes operate. In addition to their genomic plasticity, these regions are likely to have unusual chromatin structure due to their proximity to telomeric heterochromatin and abundance of other tandem repeats. We will survey the epigenetic state of many chromosomal ends, but give more attention to subtelomeres with highest relevance to human function and disease. One such region is 4qter, where recurrent contraction of a tandem array, in the context of just one of two structurally variant subtelomeric alleles (4qA), leads to facio-scapulo-humeral dystrophy (FSHD), the third most common inherited muscular dystrophy. This deletion is believed to induce epigenetic changes that cause inappropriate gene expression in 4q or elsewhere, but it is not known how this occurs or what genes are affected. We aim to identify the genetic and epigenetic features of normal and FSHD-causing 4qA alleles in order to provide a link between the 4q deletion and aberrant muscle function. This work should lead to better diagnosis and counseling of recurrence risk for FSHD patients. We will also focus on regions surrounding WASH genes, which exist in grossly different allelic and paralogous contexts in human subtelomeres. We recently discovered that WASH genes encode a highly conserved, widely expressed, yet previously unrecognized new subfamily of WASP proteins, which reorganize the actin cytoskeleton in response to extracellular signals. To accomplish our goals, we will characterize in detail genomic differences among 4qter alleles and among selected chromosomes carrying WASH, as these regions have very limited representation in the current human genome assembly. For epigenetic profiling, we will develop a microarray covering sequences in and near subtelomeres, regions lacking from other arrays. We will use these arrays to analyze subtelomeres in various cells for chromatin modifications characteristic of repressed and active chromatin. In parallel, we will use FISH to analyze larger-scale epigenetic features of specific WASH or 4q copies, such as nuclear location, chromatin condensation, and matrix association. Finally, we will apply tools capable of distinguishing SNPs and paralogous sequence variants (PSVs) to directly relate epigenetic characteristics with transcriptional activity of specific copies. This research will illuminate the significance of subtelomeric genomics and epigenetics in normal phenotypic variation among humans, as well as in inherited disorders, cancer, and aging.